Retrotransposons comprise almost half of the human genome and substantial fractions of other metazoan genomes. Nonetheless, the mechanisms and localization of retrotransposon assembly are poorly understood. Ty3 is a long terminal repeat retrotransposon in budding yeast containing GAG3 and POL3 ORFs encoding structural and catalytic proteins, respectively. Because Ty3 exists in a simple eukaryote that facilitates molecular, genetic, biochemical, and even cytological approaches, it provides a useful model for understanding retrotransposon mechanics. In the current funding period, the assembling Ty3 VLP was characterized by density sedimentation, RNA protection, transmission EM and atomic force microscopy performed on Ty3 VLPs blocked at specific stages and also on mature VLPs. In addition, sixty Ala substitution mutants were analyzed to dissect the functions of Gag3 (capsid, spacer, and nucleocapsid) subdomains. These studies showed that the amino-terminal domain is critical for assembly and nuclear pore association of VLPs and that the nucleocapsid domain is important for targeting Ty3 RNA and Gag3 protein to the P body for assembly. A set of Ty3 RNA variants was developed and used to show that the Ty3 5' and 3' untranslated regions (UTRs) and POL3 contain independent cis-acting P-body targeting activities, but that only the UTR mediates packaging of Ty3 genomic RNA. The finding that the RNA processing body (P body) previously described as a site where nontranslating RNAs are sequestered or degraded, is the site of Ty3 assembly was particularly significant as assembly sites have not been characterized for retrotransposons and how retroviruses preassemble RNA and protein at a perinuclear site is not well understood, but appears to involve packaging signals. In Aim A of the proposed work, we will use mass spectroscopy to identify host proteins which are associated with Ty3 RNA and structural protein. Our collection Ty3 wt and variant RNAs tagged with the MS2- binding site coupled with a novel high affinity TAP-tagged MS2 binding protein and our antibodies against Ty3 proteins will be used to isolate proteins directly involved with Ty3 components. Strains with attenuated functions of identified genes will be tested for defects in Ty3 VLP assembly and proteins identified will be investigated in Aims B-D. A key step in assembly occurs when the mRNA transitions from translation to packaging. Based on the retrovirus model, this is associated with structural rearrangements in the UTRs of the RNA which are facilitated by nucleocapsid binding. In Aim B we will define at higher resolution the packaging site of Ty3 RNA and its interaction with the reverse transcription primer initiator tRNAMet. Previous work in the Darlix laboratory, with whom we collaborated, showed that initiator tRNAMet has a bipartite primer binding site in Ty3 5' and 3' UTRs. Retroviruses have dimeric genomes and we have shown that Ty3 is similar. Darlix proposed a novel model for Ty3 genomic RNA dimerization mediated by two itRNAMet molecules annealing at the 5' ends. We will specifically test whether the PBS, and by implication, the putative dimerization interface, is required for packaging of Ty3 RNA into VLPs. If so, that would suggest that initiator tRNAMet, which has unique roles in translation initiation, and priming Ty3 reverse transcription, is a central player in the key transition from translation to packaging. SELEX and in vitro RNA-protein binding assays will be used to identify important nucleocapsid binding contacts in the packaging site of the Ty3 UTR which could mediate primer annealing and dimerization. In Aim C we will test our hypothesis that the Ty3 UTR structure antagonizes translation initiation and imposes requirements for specialized translation helicases. In addition, we propose that reduced numbers of translating ribosomes downstream of the frameshift site attenuates 60S subunit delivery to the 5' end of the RNA and explains the sensitivity of Ty3 and other retrotransposons to imbalances in translation initiation factors and 60S ribosomal subunits. This hypothesis will be tested using host mutants and variant Ty3 RNAs. We will test whether translation is ultimately repressed by Gag3 binding in cis to the Ty3 UTR or POL3. In Aim D we will characterize the contribution of P bodies to Ty3 assembly. We hypothesize that P-body proteins cooperate with Gag3 to repress Ty3 translation, allow the concentration of assembly factors, and may promote unwinding of the RNA to allow packaging. Nonetheless, isolation of an unusual mutant which does not form large P body clusters suggests that P bodies might repress transposition at a post-assembly stage. Factors identified in genetic and mass spectroscopy screens will be tested for direct interactions with Ty3 components and for their roles in clustering P body proteins and assembling Ty3 viruslike particles. In summary, Ty3 is a well-characterized retrotransposon in a model eukaryote. It offers a unique opportunity to understand the retrotransposon particle assembly process and how P bodies might chaperone this process.